A dual participation of ZAP-70 and scr protein tyrosine kinases is required for TCR-induced tyrosine phosphorylation of Sam68 in Jurkat T cells.
Sam68 has been initially described as a substrate of src kinases during mitosis in fibroblasts. Recent evidence suggests that in T lymphocytes Sam68 may act as an adaptor protein and participate in the early biochemical cascade triggered after CD3 stimulation. A direct interaction between Sam68 and the two src kinases involved in T cell activation, p59(fyn) and p56(lck), as well as a partnership of Sam68 with various key downstream signaling molecules, like phospholipase Cgamma-1 and Grb2, has been shown. In this study we analyze the contribution of p56(lck), as well as the role of ZAP-70, the second class of protein tyrosine kinase involved in T cell activation, in Sam68 tyrosine phosphorylation in the human Jurkat T cell line. Using the src inhibitor PP1 [4-amino-5-(4-methylphenyl)7-(t-butyl) pyrazolo [3,4-d] pyrymidine] and cell variants with defective expression of p56(lck) or expressing a dominant negative form of ZAP-70, we demonstrate that, while both p56(lck) and ZAP-70 are dispensable for the low constitutive phosphorylation of Sam68 observed in Jurkat cells, a cooperation between the two kinases is required to increase its rapid phosphorylation observed in vivo after CD3 stimulation. We also show that recombinant forms of both p56(lck) and ZAP-70 phosphorylate Sam68 in vitro. However, using CD2 stimulated cells, we observe that p56(lck) activation by itself does not induce Sam68 tyrosine phosphorylation. We conclude that p59(fyn) and p56(lck) differently participate in regulating the phosphorylation state of Sam68 in T cells and that ZAP-70 may contribute to Sam68 tyrosine phosphorylation and to the specific recruitment of this molecule after CD3 stimulation.
Lang
V
V
Laboratoire d'Immunologie Cellulaire et Tissulaire, CNRS URA 625, Centre Hospitalier Pitié-Salpêtrière, Paris, France.
Mège
D
D
Semichon
M
M
Gary-Gouy
H
H
Bismuth
G
G
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells / Q000276:immunology* / Q000378:metabolism; D010750:Phosphoproteins / Q000276:immunology* / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000276:immunology* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000276:immunology* / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000276:immunology*; D015398:Signal Transduction / Q000276:immunology*; D014443:Tyrosine / Q000378:metabolism; D051746:ZAP-70 Protein-Tyrosine Kinase; D019061:src-Family Kinases / Q000276:immunology* / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; C074561:GAP-associated protein p62; C489559:KHDRBS1 protein, human; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D011948:Receptors, Antigen, T-Cell; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; D019061:src-Family Kinases